Project Summary Zika virus (ZIKV) RNA can be detected in semen for up to 6 months post-disease onset in some men; yet, whether prolonged ZIKV RNA shedding in semen represents ZIKV sexual transmission risk is not known. This is critical to understand because sexual transmission of ZIKV from an infected man to a pregnant woman has serious risks of ZIKV congenital disease. The long-term goal of this project is to understand the mechanism of prolonged ZIKV RNA shedding in semen and the risk of sexual transmission. The objectives of this study are to identify the infectious potential of ZIKV in semen and ZIKV genetic variants associated with prolonged shedding in semen. The hypothesis is that leukocytes in semen harbor ZIKV RNA, which is no longer replication competent during prolonged ZIKV RNA shedding. Two specific aims will address this hypothesis: 1) Assess sexual transmission risk during prolonged ZIKV shedding in semen; and 2) Determine the semen- specific genetic signature of ZIKV RNA. In the first aim, semen samples from a large cohort of infected male travelers with previously-confirmed ZIKV seminal fluid shedding will be used. The localization of ZIKV RNA in human semen cells will be determined, including ZIKV RNA replication intermediates. The effect of immunosuppression on ZIKV recrudescence in semen will be tested in a mouse model. In the second aim, ZIKV genetic variants will be identified in longitudinal semen samples from the human cohort. Specific mutations previously identified during ZIKV seminal shedding in mice will be tested for sexual transmission potential in a mouse model. The research proposed here is innovative because it investigates a cell population in semen that has not been previously studied using clinical samples from a large cohort of ZIKV-infected men. Upon successful completion of the proposed research, the anticipated contribution of this work will be the identification of the cellular source of ZIKV in semen and the viral correlates of prolonged ZIKV shedding in semen. This contribution is expected to be significant because it will lead to the ability to assess and prevent the risk of ZIKV sexual transmission.